Tissue factor (TF), a transmembrane glycoprotein, is the primary cellular initiator of blood coagulation. It is predominantly expressed on the surface of sub-endothelial cells, such as smooth muscle cells and fibroblasts, and binds both zymogen coagulation Factor VII (FVII) and the activated form, Factor VIIa (FVIIa) when the integrity of the endothelium is interrupted, such as when blood vessels are severed. When TF binds FVII, it promotes FVII to FVIIa activation. TF also greatly enhances the proteolytic activity of Factor VIIa towards its physiologic substrates, Factors IX and X. FVIIa retains a zymogen-like state in solution, acting as a relatively poor enzyme. TF provides a scaffold for optimal macromolecular exosite interaction, induces conformational changes in the protease domain of FVIIa leading to maturation of its active site and orientates FVIIa on a cell surface for optimal substrate interaction. Together these effects results in an enhanced catalytic capability of FVIIa of several orders of magnitude. Hence, TF is a co-factor for FVIIa in the initiation complex of what is traditionally referred to as the extrinsic pathway of blood coagulation. Subsequent steps of the coagulation cascade finally result in the formation of a fibrin polymer which is bound by activated platelets and crosslinked with FXIIIa.
Platelets—also known as thrombocytes—derive from their cellular predecessor, megakaryocytes. Normal resting platelets freely flow throughout the blood circulation when the endothelium is intact. When the single-layered endothelial barrier is damaged, resting platelets adhere to subendothelial structures by means of glycoprotein (GP) receptors. For example, GPIaIIa and GPVI bind collagen; GPIcIIa binds fibronectin; GPIc*IIa binds laminin and GPIb-V-IX binds von Willebrand Factor (vWF) polymers. Adhesion of platelets in this manner causes them to change shape and release their alpha and dense granules. In turn, this results in the exposure of a plethora of other glycoprotein platelet receptors, such as GPIIbIIIa (which binds fibrinogen/fibrin) and TREM-like transcript 1 (TLT-1); as well as the release of coagulation factors I (fibrinogen), V and XI; other procoagulants such as ADP, Ca2+, serotonin and Platelet Factors 3/4; anti-coagulants such as tissue factor pathway inhibitor (TFPI); and compounds such as platelet derived growth factor (PDGF), essential to platelet replenishment and healing. Activated platelets bind to one other and cross-link fibrin in a rapid reaction for example via the GPIIbIIIa receptor complex.
Hence, activated platelets and the fibrin polymer product of the coagulation cascade together form the blood clot. Platelet aggregation of coagulation is known as the primary hemostatic response, while the coagulation cascade response is known as the secondary hemostatic response. Although fibrin is produced during the primary hemostatic response, via the so-called coagulation initiation (independent of FIX/FVIIIa), the amount produced at this point is insufficient for a strong coagel. Initial fibrin serves as an aggregater of activated platelets at site of injury, which again provides an optimal cell surface for the function of activated coagulation factors.
In subjects with a coagulopathy, such as in human beings with haemophilia A and B, various steps of the coagulation cascade are rendered dysfunctional due to, for example, the absence or insufficient presence of a coagulation factor. Such dysfunction of one part of coagulation results in insufficient blood coagulation and potentially life-threatening bleeding.
An object of the current invention is to provide a compound that is suitable for use as a procoagulant drug in such subjects. A second object of the current invention is to provide a compound that enables a physical point of initiation of blood coagulation to be mobilised, such that extrinsic coagulation is not solely dependent on subendothelial, cell-bound tissue factor. A third object of the current invention is to provide a compound that up-regulates blood coagulation in a physiologically suitable microenvironment. A further object of the current invention is to direct a soluble tissue factor, or a biologically functional fragment or variant thereof, to the surface of activated platelets. A further object of the invention is to enhance the proteolytic activity of endogenous Factor VIIa towards its physiological substrates, Factors IX and X. Thus, the object is to enable the initiation of blood coagulation on the surface of activated platelets that are located intravascularly or extravascularly. This is in addition to the normal and exclusively subendothelial—typically extravascular—initiation of blood coagulation.
WO06/096828 discloses chimeric proteins that comprise soluble tissue factor (sTF) and a phosphatidyl serine (PS) binding domain, such as Annexin V. PS is exposed on the surface of activated cells, such as monocytes, endothelial cells and cells undergoing apoptosis, as well as on activated and resting platelets. The chimeric proteins are both pro-coagulant and anti-coagulant; the latter due to the fact that, in higher doses, constructs compete with coagulation factors in binding to PS on activated platelets. Thus, the chimeric proteins of WO06/096828 have a different set of properties than the fusion proteins described herein.